W. Scott Grayburn

FvVE1 regulates filamentous growth, the ratio of microconidia to macroconidia and cell wall formation in Fusarium verticillioides

The velvet gene, veA, co‐ordinates asexual and sexual development in the homothallic fungal species Aspergillus nidulans. Studies in Aspergillus parasiticus and Aspergillus fumigatus demonstrated that veA also regulates morphological differentiation in these species. Whether veA has the same role in morphogenesis in other fungal genera has not been investigated. In this work, we studied the role of the veA homologue, FvVE1, in the heterothallic fungus Fusarium verticillioides. Deletion of FvVE1 suppressed aerial hyphal growth and reduced colony surface hydrophobicity on solid media. In submerged cultures, FvVE1 deletion caused alterations in hyphal polarity, marked activation of conidiation and yeast‐like growth. The latter was promoted by shaking to increase aeration of cultures. In addition, FvVE1 deletion markedly increased the ratio of macroconidia to microconidia. Supplementation of osmotic stabilizers restored the wild‐type phenotype to deletion mutants, suggesting phenotypic alterations caused by FvVE1 deletion are related to cell wall defects. This is consistent with the hypersensitivity of FvVE1 deletion mutants to SDS and with the significant reduction in the mannoprotein content of mutants compared with the wild‐type strain. However, no dramatic cell wall alterations were observed when mutants were examined by transmission electron microscopy. Our data strongly suggest that FvVE1 is important for cell wall integrity, cell surface hydrophobicity, hyphal polarity and conidiation pattern.

Non-DNA-binding platinum anticancer agents: Cytotoxic activities of platinum–phosphato complexes towards human ovarian cancer cells

DNA is believed to be the molecular target for the cytotoxic activities of platinum (Pt) anticancer drugs. We report here a class of platinum(II)- and platinum(IV)-pyrophosphato complexes that exhibit cytotoxicity comparable with and, in some cases, better than cisplatin in ovarian cell lines (A2780, A2780/C30, and CHO), yet they do not show any evidence of covalent binding to DNA. Moreover, some of these compounds are quite effective in cisplatin- and carboplatin-resistant cell line A2780/C30. The lack of DNA binding was demonstrated by the absence of a detectable Pt signal by atomic absorption spectroscopy using isolated DNA from human ovarian cells treated with a platinum(II)-pyrophosphato complex, (trans-1,2-cyclohexanediamine)(dihydrogen pyrophosphato) platinum(II), (pyrodach-2) and from NMR experiments using a variety of nucleotides including single- and double-stranded DNA. Furthermore, pyrodach-2 exhibited reduced cellular accumulations compared with cisplatin in cisplatin- and carboplatin-resistant human ovarian cells, yet the IC50 value for the pyrophosphato complex was much less than that of cisplatin. Moreover, unlike cisplatin, pyrodach-2 treated cells overexpressed fas and fas-related transcription factors and some proapoptotic genes such as Bak and Bax. Data presented in this report collectively indicate that pyrodach-2 follows different cytotoxic mechanisms than does cisplatin. Unlike cisplatin, pyrodach-2 does not undergo aquation during 1 week and is quite soluble and stable in aqueous solutions. Results presented in this article represent a clear paradigm shift not only in expanding the molecular targets for Pt anticancer drugs but also in strategic development for more effective anticancer drugs.

Secondary metabolism and development is mediated by LlmF control of VeA subcellular localization in Aspergillus nidulans.

Secondary metabolism and development are linked in Aspergillus through the conserved regulatory velvet complex composed of VeA, VelB, and LaeA. The founding member of the velvet complex, VeA, shuttles between the cytoplasm and nucleus in response to alterations in light. Here we describe a new interaction partner of VeA identified through a reverse genetics screen looking for LaeA-like methyltransferases in Aspergillus nidulans. One of the putative LaeA-like methyltransferases identified, LlmF, is a negative regulator of sterigmatocystin production and sexual development. LlmF interacts directly with VeA and the repressive function of LlmF is mediated by influencing the localization of VeA, as over-expression of llmF decreases the nuclear to cytoplasmic ratio of VeA while deletion of llmF results in an increased nuclear accumulation of VeA. We show that the methyltransferase domain of LlmF is required for function; however, LlmF does not directly methylate VeA in vitro. This study identifies a new interaction partner for VeA and highlights the importance of cellular compartmentalization of VeA for regulation of development and secondary metabolism.

Aspergillus nidulans Natural Product Biosynthesis Is Regulated by MpkB, a Putative Pheromone Response Mitogen-Activated Protein Kinase

ABSTRACT The Aspergillus nidulans putative mitogen-activated protein kinase encoded by mpkB has a role in natural product biosynthesis. An mpkB mutant exhibited a decrease in sterigmatocystin gene expression and low mycotoxin levels. The mutation also affected the expression of genes involved in penicillin and terrequinone A synthesis. mpkB was necessary for normal expression of laeA, which has been found to regulate secondary metabolism gene clusters.

Complex Transitions between C3 and C4 Photosynthesis during the Evolution of Paniceae: A Phylogenetic Case Study Emphasizing the Position of Steinchisma hians (Poaceae), a C3‐C4 Intermediate

A two‐tiered, nested molecular phylogenetic study of panicoid grasses to explore character state transitions between the C3 and C4 adaptive syndromes is presented. A broad survey of 92 panicoid species was sampled for the grass‐specific insert sequence in the chloroplast RNA polymerase locus (rpoC2), combining published and unpublished sequences. This portion of the study also included an intensive phylogenetic investigation of one clade of seven species that included Steinchisma hians, which is notable for exhibiting intermediacy between the C3 and C4 photosynthetic types. Both rpoC2 data and previously published sequences of the F subunit of an NADH‐dependent dehydrogenase were analyzed together for this small group. A rigorous phylogenetic investigation of S. hians and 13 other species of Panicoideae included in the broad survey was then performed with sequences of both rpoC2 and the externally transcribed spacer region of the nuclear ribosomal repeat. These 14 species were selected to maximize representation among photosynthetic subtypes. Combined analysis resolved single origins of two photosynthetic subtypes. A reversion of C4 to C3 photosynthesis during the evolution of the lineage that includes S. hians is identified. These and other recent results indicate that repeated reversions from C4 to C3 have occurred. The C3 species Panicum laxum has a strongly supported sister group relationship to S. hians (C3‐C4). The most parsimonious interpretation is that S. hians represents an incipient reversal from C3 to C4 photosynthesis, beginning with the capacity to compartmentalize photorespiratory metabolism in the bundle sheath tissue.

Role of Nitric Oxide and Flavohemoglobin Homolog Genes in Aspergillus nidulans Sexual Development and Mycotoxin Production

ABSTRACT Flavohemoglobins are widely distributed in both prokaryotes and eukaryotes. These proteins are involved in reducing nitric oxide levels. Deletion of the Aspergillus nidulans flavohemoglobin gene fhbA induced sexual development and decreased sterigmatocystin production. Supplementation with a nitric oxide-releasing compound promoted cleistothecial formation and increased nsdD and steA expression, indicating that nitric oxide induces sexual development. This is the first study on the effect of nitric oxide on morphogenesis and secondary metabolism in fungi.

Variable abundance of a mitochondrial DNA fragment in cultured tobacco cells.

SummaryThe relative abundance of a cloned 4.5 kilobase (kb) pair mitochondrial DNA sequence in two suspension cultures of tobacco (Nicotiana tabacum cv Turkish samsun and Nicotiana tabacum NT-1) has been examined. This sequence is 70-fold reduced in NT-1 relative to Turkish samsun; the reduction is correlated with an increase in supercoiled mitochondrial DNA. This sequence does not hybridize with mitochondrial DNA from watermelon, maize, or Saccharomyces cerevisiae, nor with several cloned mitochondrial genes and is thus probably not a gene. It may represent most of the plant mitochondria) genome thought to be non-essential for mitochondrial function. The sequence complexity of supercoiled mitochondrial DNA from NT-1 cells is about one-third that found for the entire mitochondrial genome and does not include the cytochrome oxidase subunit II gene.

The mitochondrial genome of Saprolegnia ferax: organization, gene content and nucleotide sequence

The mitochondrial genome of the peronosporomycete water mold Saprolegnia ferax has been characterized as a 46 930 bp circle containing an 8618 bp large inverted repeat (LIR). Eighteen reading frames encode identified subunits of respiratory complexes I, III, IV and V; 16 encode polypeptides of small and large mitoribosome subunits; and one encodes a subunit of the sec-independent protein translocation pathway. Of four additional putative reading frames three are homologues of those found in the related Phytophthora infestans genome. Protein encoding loci in the tightly compacted genome typically are arranged in operon-like clusters including three abutting and two overlapping pairs of reading frames. Translational RNAs include the mitochondrial small and large subunit rRNAs and 25 tRNA species. No tRNAs are encoded to enable translation of any threonine or the arginine CGR codons. The LIR separates the molecule into 19 274 bp large and 10 420 bp small single copy regions, and it encodes intact duplicate copies of four reading frames encoding known proteins, both rRNAs, and five tRNAs. Partial 3′ sequences of three additional reading frames are duplicated at single copy sequence junctions. Active recombination between LIR elements generates two distinctive gene orders and uses the duplicated 3′ sequences to maintain intact copies of the partially duplicated loci.